To study the role of cellular sulfation in embryonic brain development, the investigators have developed a novel inducible RNAi system to reduce the cellular levels of PAPS, the universal sulfate donor in the cell. They have generated transgenic mice which can be induced through Cre-mediated recombination to express an RNAi hairpin targeting one of two PAPS synthesizing enzymes, PAPSS1, which is abundantly expressed in brain during neurogenesis. Crossing the inducible PAPSS1 RNAi mice to Nestin-Cre mice result in expression of the RNAi hairpin in neural progenitor cells beginning at approximately E9.5. Mice from these matings survive to adulthood but have brains that are markedly smaller (microcephaly) than control littermates. Preliminary characterization of the PAPSS1 RNAi brain reveals reduced PAPS synthetase activity and a narrow window of apoptosis early in neurogenesis. The cause of the apoptosis event is unknown, though the investigators have evidence that it may be due to oxidative stress. The objective of this application is to begin to elucidate the mechanism by which cellular sulfation influences cell survival in early brain development. Towards this goal, the investigators will first characterize the PAPSS1 brain specific knock-down phenotype by examining total PAPSS activity, cell proliferation, cell death, and laminar patterning. Second, to explore the cause of cortical cell death in the PAPSS1 RNAi mouse and how it might lead to the observed reduction in brain size, they will analyze indicators of oxidative stress and formation of reactive oxygen species (ROS), as well as determine whether oxidative stress may be causing DNA damage in the PAPSS1 knock-down. Furthermore, changes in extracellular proteoglycan sulfation (a major use of the PAPS generated by PAPS synthetase) will be assessed to determine whether proteoglycan sulfation can affect ROS levels during neuronal differentiation. The proposed studies will provide a comprehensive understanding of the phenotypic consequences of reduced PAPSS1 expression, and a substantial foundation for subsequent investigation into the roles of sulfation in the developing brain. PROJECT NARRATIVE: Malformations arising during cortical development are increasingly recognized as important causes of epilepsy and developmental delay. This application makes use of mouse models to elucidate the function of sulfation during brain cortical development. Since smaller brain size is the main phenotype in the proposed transgenic animals, the investigators anticipate that these studies will lead to a better understanding of microcephaly in humans.